Periodontitis, an inflammatory disease of tissues in the subgingival crevice, is associated with a dramatic shift in the subgingival microflora towards predominantly gram negative organisms. To understand why certain bacteria are successful pathogens, it is important to determine the structure, function, and regulation of each of their virulence factors. Actinobacillus actinomycetemcomitans has been strongly implicated in Localized Juvenile Periodontitis and in several adult periodontal disorders. The primary candidate virulence factor produced by A. actinomycetemcomitans is a leukotoxin which kills host neutrophils. Bacterial isolates associated with pathogenesis usually produce high levels of leukotoxin. Moreover, the levels of this virulence factor are regulated by changes in the environment; both anaerobic and high salt conditions favor increased production of leukotoxin. We now propose to exploit the molecular genetic tools we have developed in order to determine the mechanisms of strain-specific and environmental regulation of the leukotoxin. Both genetic (transposon mutagenesis, complementation and gene-deletion) and biochemical (gel mobility shift, affinity chromatography) approaches will be used to identify the leukotoxin promoter sequences critical in regulation and to characterize the proteins that are involved. The genes encoding these regulatory proteins will be cloned and used to generate specific mutations in the endogenous A. actinomycetemcomitans genes. The resulting mutants, deficient in various leukotoxin regulatory pathways, will be tested for defects in general and in environmental regulation. Importantly, these mutant strains of A. actinomycetemcomitans can be used in animal models in future studies to assess the role of individual regulatory pathways in the pathogenesis of A. actinomycetemcomitans. Knowledge of the mechanisms of regulation of virulence factors may lead to the future design of novel drugs that target transcription factors involved in regulating several different virulence components.